The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.